Application of additively manufactured 3D scaffolds for bone cancer treatment: a review

Bone cancer is a critical health problem on a global scale, and the associated huge clinical and economic burdens are still rising. Although many clinical approaches are currently used for bone cancer treatment, these methods usually affect the normal body functions and thus present significant limitations. Meanwhile, advanced materials and additive manufacturing have opened up promising avenues for the development of new strategies targeting both bone cancer treatment and post-treatment bone regeneration. This paper presents a comprehensive review of bone cancer and its current treatment methods, particularly focusing on a number of advanced strategies such as scaffolds based on advanced functional materials, drug-loaded scaffolds, and scaffolds for photothermal/magnetothermal therapy. Finally, the main research challenges and future perspectives are elaborated.


Introduction
According to the World Health Organization (WHO), cancer caused 10 million deaths in 2020 with 19.3 million new cases detected in the same year [1]. The total annual cancer-related economic costs in the USA added up to $183 billion for 2015 [2], and e199 billion in Europe for 2018 [3]. Bone cancer is caused by cancer cells originating in bone tissue (primary bone cancer) or metastasized from other primary tumor areas to bone tissue (metastatic bone cancer) [4]. Around 700,000 new primary bone cancer cases are reported each year worldwide [5,6]. Due to the low incidence of bone cancer, its delayed diagnosis and treatment are common, leading to high mortality. More than 220,000 patients die following treatment each year, resulting in a global survival rate of around and gold) is a viable approach to overcome these limitations [20,21]. Other strategies are aimed at releasing therapeutics near the cancer regions in response to environmental changes (e.g., temperature or pH value), thus reducing the risks of using high doses [20]. However, therapeutics still enter the systemic circulation, and the clinical problems related to large tissue defects following tumor resections remain [22].
Since the 1970s, different strategies have been proposed in combination with the use of implants, including delivering antibiotics to prevent bacterial infections and biofilm formation [23,24], and adding molecules such as growth factors [25,26]. Clinical implants can also be loaded with chemotherapeutics for local cancer treatment applications, combining the advantages of post-operative clinical implants and chemotherapy [27,28]. One of the first such examples is Gliadel ® Wafer (Eisai Inc., Japan), a drug-loaded implant for malignant glioma treatment [29]. Implants made from functional materials and those containing chemotherapeutics and targeted cancer drugs, implanted in the resected cancer region, represent a novel and promising approach. In this case, the implant should not only provide mechanical support, but also promote tissue repair, prevent cancer recurrence, and improve patient outcomes [30]. Another strategy is based on the use of implants sensitive to external electromagnetic radiation for photothermal/magnetothermal therapy. These implants contain photo-sensitizers or magnetosensitizers; they are placed on the site of the lesion, excited with a near-infrared (NIR) radiation or a magnetic field, and, as a consequence, release heat. As cancer cells have a lower heat tolerance than normal cells, the induced heat from the implants leads to irreversible protein denaturation, cell membrane damage, and progressive apoptosis [31]. Contrary to conventional treatments, local photothermal/magnetothermal therapies can selectively and effectively eliminate cancer cells without damaging the surrounding normal cells [32]. All of these approaches have been considered for the treatment of both primary bone cancer and/or metastatic bone cancer.
Due to the limitations of conventional bone cancer therapies, new and more effective methods are being investigated, and in turn several advanced approaches have been proposed using implants for local sustained treatment and photothermal/magnetothermal therapy. These implants are fabricated using additive manufacturing to achieve high customization, high reproducibility, and relatively low fabrication costs. These novel therapeutic methods seem to represent suitable approaches for future bone cancer treatment and post-treatment bone regeneration. This review discusses the limitations of the current clinical treatment methods of bone cancer and provides a detailed review of novel treatment approaches using additively manufactured 3D clinical implants as local cancer treatment substrates. The key research challenges are also discussed.

Bone cancer and corresponding treatments
Bone makes up the human skeleton, which supports, moves, and protects the human body and the internal organs. It also provides stable points for muscles to attach, stores essential nutrients and minerals critical for different body functions [33], and produces lipids and blood cells [34]. Bone cells (osteoblasts, osteocytes, and osteoclasts) are responsible for new bone formation, mineralization, and bone destruction [35,36]. As a load-bearing tissue, the bone is continuously exposed to shear and compressive forces, which further modulate bone homeostasis (Fig. 1a) [37]. The connection between the bone niche and the etiology of primary bone cancer has not been clearly defined. However, research has indicated that some acquired and congenital factors can increase the incidence of primary bone cancer, and regular inspections are critical to prevent the onset of bone cancer [37].

Primary bone cancer
According to the WHO, primary bone cancers can be classified into chondrogenic tumors, miscellaneous tumors, notochordal tumors, osteogenic tumors, vascular tumors, and other malignant mesenchymal tumors [38].
Osteosarcoma is one of the most common of primary bone cancers, with around 150 new cases each year in the UK [39]. The incidence ratio between males and females is 1.4:1 and the two age groups with peak incidence are adolescents aged 10-19 years and adults above 60 years of age [40,41]. Osteosarcoma is more common in adolescence in Afro-Caribbeans and Caucasian adults [42]. Younger patients have a significantly higher 5-year survival rate than patients aged over 40 (53% in the case of patients below 40, and 22% in the case of patients over 40) [43]. The high-incidence regions include the metaphysis of long bones such as the ankle and knee joint, affecting the femur (40%, usually distal), tibia (20%), humerus (10%), and pelvis (8%) [42]. The etiology of osteosarcoma is still unclear. However, it is widely accepted that loading stresses and rapid bone growth, which can be attributed to the pubertal growth spurt and regions in the metaphysis of long bones, cause DNA mutations leading to disease [12,44].
Ewing sarcoma, another common primary bone cancer, exhibits different degrees of neuroectodermal differentiation [45]. It has higher incidence in males than females (1.5:1) [46]. Around 80% of Ewing sarcoma patients are children and adolescents, with a median age of around 15 years [12]. Whites have a higher incidence rate than Asians and Blacks [12]. Its survival rate highly depends on the metastasis status, with around 20-25% of diagnosed cases showing clinically Fig. 1 a Cell-cell and cell-matrix interactions in a multifactorial bone cancer niche (Reproduced from [37], Copyright 2019, with permission from Elsevier). The mechanism of b normal bone remodeling, c oste-olytic bone metastasis, d osteoblastic bone metastasis, and e vicious cycle (Reproduced from [56], Copyright 2016, with permission from Springer Nature) detectable metastasis. In the case of localized tumors, the 5-year survival rate is around 50-60%, and it significantly decreases to around 20% in the case of metastatic sarcoma [47]. Approximately 60% of Ewing sarcoma cases start from flat bones and 40% of them affect the metaphysis of long bones [45,48]. This type of primary bone cancer typically occurs in the legs, pelvis, ribs, and vertebral column [12]. Ewing sarcoma is a non-complex karyotype sarcoma, as specific gene fusion proteins (e.g., EWS/FLI1) drive tumor oncogenesis [7,42]. It can be characterized by a variety of chromosomal translocations, but 85-90% of cases correspond to the t(11;22)(q24;q12) chromosomal translocation, involving the Ewing sarcoma gene on chromosome 22 and the Friend leukemia virus integration site 1 (FLI1) gene on chromosome 11 [44,49].
Due to the ageing population, chondrosarcoma is the most common primary bone cancer in many countries, with around 180 new cases in the UK each year [44]. There are no significant differences in the incidence between genders and ethnicities, while adults aged between 30 to 60 have a higher risk of incidence [41]. Long bones, such as pelvis and ribs (45%), ilium (20%), femur (15%), and humerus (10%), have a higher incidence risk than flat bones like the scapula [50]. Similar to other bone cancers, chondrosarcoma can be characterized by marked heterogeneity at the histological, genetic, and epigenetic levels [7].

Metastatic bone cancer
Bone tissue is one of the most frequent sites of cancer metastasis, third to the lung and the liver [51]. Metastatic bone cancer is usually manifested in severe pain, impaired mobility, pathologic fractures, spinal cord compression, bone marrow aplasia, and hypercalcemia [52]. Cancer cells from the primary cancer regions can be relocated through the bloodstream and the lymphatic system to the bone. Briefly, the metastasis of cancer cells includes the loss of intercellular cohesion, cell migration, angiogenesis, access to systemic circulation, survival in circulation, the evasion of local immune responses, and growth at distant organs [53,54]. Vascular adhesion and extravasation, microenvironmental support, and epithelial-mesenchymal transition are the critical factors affecting metastasis [51]. The metastasis of cancer to bone usually means short-term prognosis for patients, indicating that cancer can be rarely cured, whereas the growth process can often be slowed down (Table  1).

Current bone cancer treatment approaches
Depending on the type, location and extension of cancer and the overall health condition of patients, the current clinical treatment strategies of bone cancer comprise surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, and the combinations of these methods (Table 2). Unlike primary bone cancer that usually has defined treatment guidelines [12], the treatment of metastatic bone cancer generally requires case-by-case consultation by doctors. All of the different treatment approaches may be applied depending on the type of primary tumor, the bones that primary cancer has invaded, the extent of damage to the bones, the types of treatment already applied, and the overall health conditions of affected patients [65]. As cancer metastasis to bone usually corresponds to terminal cancer stages and short-term prognosis, treatments focus on pain relief, preventing complications and improving the life quality [65].

Surgery
Surgery, an important bone cancer treatment method, is usually combined with other approaches [67]. The primary goal of surgery is to completely resect the cancer region, and the secondary goal is to keep the limb as functional as possible. Therefore, limb-sparing surgery (LSS) is always the first approach considered to avoid completely removing an affected body part. If the tumor located on the bone does not affect the body function, such as ribs, the bones can be directly removed. In other cases, tumors will be removed and replaced by bones from a bone bank, autologous bones, or an artificial implant [12,67]. In these cases, the surgical area is often larger than the cancer region to achieve the full removal of the tumor and to prevent its recurrence [68]. In some rare cases (e.g., extracorporeal irradiation), bones will be taken out, irradiated, and returned to the patient's body [12]. Although the LSS procedure allows for better functional results than amputation, up to 10% of patients still need to have a limb permanently removed (e.g., in cases of large tumors size, critical nerve involvement, and complicated cancer region locations) [67,69].
As the growth of cancer region is a process occurring from inside out, the surrounding tissue is compressed and forms a pseudocapsule at the interface between the cancer region and the normal tissue, and pseudopods of the tumor extend into this interface [70]. Thus, an adequate surgical margin (the boundary of removed tissue) is critical to prevent bone cancer recurrence [71]. Enneking [72,73] described four types of surgical margin (Table 3), and the National Comprehensive Cancer Network guidelines recommend the wide local excision of bone sarcomas. Moreover, resection is often more challenging in the pelvis and spine than it is in the extremities due to the associated anatomical complexity [74]. Although LSS may allow for better functional results than amputation, the risk of post-surgical complications is three times higher, and the risk of complications after end prosthetic reconstruction is fourfold [75]. These complications can be classified as mechanical (e.g., soft tissue failure, aseptic loosening, and breakage/fracture/dislocation of the implant), non-mechanical (e.g., infection and tumor progression) and pediatric (e.g., growth arrest and joint dysplasia) [76].

Chemotherapy
Chemotherapy is an approach that uses cytotoxic drugs to suppress cancer cells to prevent them from growing and spreading in the body, and to reduce the risk of recurrence of cancer [67]. This clinical approach is mainly performed via oral and intravenous administration, while other administration methods have been also used, such as subcutaneous, intramuscular, intrathecal, and the use of skin creams [67,77,78]. Chemotherapy is usually combined with other treatments and can be used before surgery (neo-adjuvant chemotherapy) to decrease the risk of distant recurrence after surgery or to control the development of primary cancer [67,78]. This treatment can also be carried out after radiotherapy or surgery (post-operative adjuvant chemotherapy) to reduce the risk of cancer recurrence [67,78]. It can also be used individually to relieve symptoms in cases where it is impossible to fully cure the patient (palliative chemotherapy) [67,78]. Chemotherapy is one of the most frequently used treatment approaches for non-metastatic high-grade osteosarcomas such as Ewing sarcoma, which includes treatments with neo-adjuvant chemotherapy followed by definitive local therapy and then adjuvant chemotherapy [77,79]. However, chemotherapy is still not fully effective for other types of bone cancer such as chondrosarcoma [12,80].
The side effects of chemotherapy can differ from patient to patient, but usually include nausea, vomiting, constipation, indigestion, insomnia, headache, anemia (by reducing the number of red blood cells that carry oxygen around the body), bruising and bleeding (by lowering the number of platelets in the blood), skin and nail changes (e.g., dryness, slight discoloration, increased sensitivity to sunlight, redness, soreness and itchyness), fatigue, hair loss, lower resistance to infections, sore mouth, loss of appetite, memory and concentration problems, sex and fertility issues [12,[80][81][82]. Moreover, chemotherapy presents other limitations such as difficulty of reaching cancer cells far from tumor vessels, and short halflife reducing drug bioavailability and treatment effectiveness [83][84][85][86].

Radiotherapy
Radiotherapy uses radiation to treat cancer and to reduce the risk of recurrence either in the early stage or after spreading. This treatment type is usually carried out by applying external ionizing radiation (radioactive beams at the cancer regions), radiotherapy implants (brachytherapy, implanting small pieces of radioactive metal near the cancer regions), radiotherapy injections, capsules or drinks (radioisotope therapy, oral or injected radioactive liquids), and intrabeam radiotherapy [67]. Advanced radiotherapy techniques, such as carbon ion beam radiotherapy, intraoperative radiation, and proton beam radiation, have also been proposed [87][88][89]. Radiotherapy can also be combined with chemotherapy or used before surgery (neo-adjuvant radiotherapy) to help reduce the cancer region and minimize the surgery area [67,90]. Moreover, it can be applied after surgery to eliminate residual cancer cells, preventing recurrence [67,90]. In the case of unresectable tumors or symptomatic widespread metastatic primary tumors, radiotherapy is used to relieve symptoms if full cure is not possible (palliative radiotherapy) [67,90]. In other cases, radiotherapy can be considered as an adjunct therapy (marginal resection), or as an alternative to surgery (e.g., in difficult locations such as pelvis, spine, and chest wall) [80]. Radiotherapy is an effective method for the local control of Ewing sarcoma, but as a standalone option is not the ideal modality for primarily localized bone sarcomas [12].
The side effects of radiotherapy include sore skin (darker than normal or dry and itchy), hair loss (unlike chemotherapy, only affecting the area being treated), eating and drinking problems (e.g., sore mouth, loss of appetite, weight loss, and discomfort when swallowing), fatigue, feeling sick, diarrhea, stiff joints and muscles, sex and fertility issues, wound complications, limb-length discrepancies, joint contractures, pathologic fractures, and secondary malignancy [12]. Moreover, it was reported that even relatively low doses of radiation could increase the incidence of sarcomas induced by radiation, usually presenting a worse prognosis than those not related to radiation exposure [91,92].

Targeted therapy
Targeted therapy inhibits the growth and spread of cancer cells by aiming at specific factors necessary for cancer development (e.g., molecules, genes, and proteins) while limiting the damage to healthy cells [4,93,94]. The target drugs can be classified into monoclonal antibodies, cancer growth blockers, antiangiogenics, and poly (ADP-ribose) polymerase inhibitors, and the common drugs include Avastin (Roche, Switzerland), Herceptin (Roche, Switzerland), Mabthera (Roche, Switzerland), Olaparib (AstraZeneca, UK), Mifamurtide (Takeda, Japan), and Imatinib (Novartis, Switzerland) [94][95][96]. Some studies have already validated targeted therapy as an effective method for treating primary bone cancer such as chondrosarcoma and chordoma [94,96] and metastatic bone cancer secondary to breast and prostate cancers [97,98]. However, due to the high heterogeneity of bone cancer, its targeted therapy is not as effective as surgery, chemotherapy, or radiotherapy [99,100]. Therefore, despite some promising results, this therapy is still not the first choice of clinicians.
The most common side effects of targeted therapy include fatigue, nausea, dizziness, vomiting, increased liver enzymes, cough, constipation, and diarrhea [101].

Immunotherapy
Immunotherapy approaches include cancer vaccines, cellbased immunotherapy, immune checkpoint blockade, and cytokines [102][103][104]. Cancer vaccines, one of the first immunotherapies to be used for bone cancer treatment [105], use neoantigens derived from whole tumor cells, tumor cell lysates, and cancer-related peptides [106,107]. Tumor antigen-specific T cells are activated by the major histocompatibility complex molecules of antigen-presenting cells and eventually trigger immune response [108]. Cell-based immunotherapy mainly uses tumor-infiltrating lymphocytes or cytotoxic T lymphocytes [107]. Cells are sorted from a patient, expanded and stimulated ex vivo, and transferred back to the patient [109]. Immune checkpoint blockade removes the brakes of the immune system [110]. The currently approved checkpoint blockade inhibits PD-1, CTLA-4, and PD-L1 [102]. Cytokines (e.g., interleukin-2 and interferons), the proteins that regulate the immune system, have been used for the immunotherapy of sarcomas [111]. Immunotherapy represents a clinically accepted approach to treat certain cancers (e.g., melanoma, renal cell carcinoma, prostate cancer, and lung cancer), but their limited success has been reported for bone cancer [103]. The possible reasons can be the relatively low immunogenicity of bone cancer [112,113], difficulty in establishing bone cancer cell lines and autologous CTL [114,115], the lack of targetable neoantigens and suitable candidate genes for a reverse immunological approach [116][117][118], and also that most bone cancer cells express immune checkpoint proteins contributing to their immune escape [118]. Further investigations of the bone cancer microenvironment and immunological milieu are needed to better understand the markers that predict the immune responsiveness of patients with sarcomas [103].
The main side effects of immunotherapy include diarrhea, rashes, nausea, vomiting, respiratory symptoms, and prolonged times to achieve effectiveness [102].

Advanced cancer treatment strategies based on synthetic 3D scaffolds
Advanced manufacturing technologies and novel materials opened up new routes for the development of new therapeutic strategies, namely the use of 3D scaffolds based on functional materials, drug delivery carriers, and scaffolds used as photothermal/magnetothermal therapy substrates. A particularly relevant method is the use of additive manufacturing (also known as 3D printing), a group of techniques that produce scaffolds by adding materials usually layer-by-layer, which offer significant advantages for the fabrication of these novel scaffolds (e.g., low cost, high accuracy and high repeatability) for both primary bone cancer and metastatic bone cancer applications.

Scaffolds with advanced functional materials
Recently, researchers developed 3D-printed scaffolds incorporating different functional materials (instead of conventional chemotherapeutics or anticancer drugs). Hou et al. [119,120] proposed the concept of dual-functional scaffolds (Figs. 2a and 2b) for bone cancer treatment and subsequent bone regeneration using graphene and graphene oxide (GO) fillers (up to 9 wt%). The first function (treatment) aims to induce cancer cell death, while the second function (regeneration) targets the induction of new bone formation. The poly(ε-caprolactone) (PCL)/graphene and PCL/GO scaffolds were produced using a screw-assisted extrusionbased additive manufacturing system. The results of in vitro cell experiments conducted with human adipose-derived stem cells (hADSCs) and sarcoma osteogenic (Saos-2) cells showed that low concentrations of graphene could slightly promote the viability of hADSCs, while high concentrations could inhibit the viability of both hADSCs and Saos-2 cells (Figs. 2c and 2d). The findings also revealed that PCL scaffolds containing high concentrations of graphene exhibit a stronger inhibition effect on Saos-2 cells than on hADSCs. It was also observed that low concentrations of GO inhibit the viability of hADSCs (Fig. 2c). It also seemed that GO had greater inhibition ability on cell viability than graphene. The a1 Bone with a cancer region is detected. a2 Bone with possible residual cancer cells (red) after tumor resection. a3 Treatment effect of the implanted dual-functional scaffold: the initial layers of scaffold containing a cytotoxic material will induce the death of possible remaining cancer cells in the surrounding tissue. a4 Regeneration phase of the implanted dual-functional scaffold: after a certain period, the layers responsible for the treatment phase disappeared (degradation mechanism), and the layers of biocompatible materials will start recruiting healthy cells promoting cell attachment, proliferation, and differentiation, inducing new bone formation. a5 Bone with regenerated tissue. b Different views of the dual-functional scaffolds, where the outer layers (black) are responsible for the treatment function and the inner layers (white) are responsible for the regeneration function. c Viability of hAD-SCs after 1, 3, and 7 days of cell seeding on PCL scaffolds, 1 wt% G scaffolds, 2 wt% G scaffolds, 3 wt% G scaffolds, 1 wt% GO scaffolds, 2 wt% GO scaffolds, and 3 wt% GO scaffolds. The percentage of surviving (d1) hADSCs and (d2) Saos-2 cells after 1, 3, and 7 days of cell seeding on 5 wt% G scaffolds, 7 wt% G scaffolds, and 9 wt% G scaffolds, with PCL scaffold as the reference (Reproduced from [119], Copyright 2020, with permission from Whioce. Reproduced from [120], Copyright 2020, with permission from Mary Ann Liebert). Viability of e MG-63 cells and f MC3T3 cells after 1, 3, and 5 days of cell seeding on GG/GO-R scaffolds, GG/GO/Cur-R scaffolds, GG/GO-H scaffolds, and GG/GO/Cur-H scaffolds. R denotes linear lattice, and H denotes honeycomb filling pattern (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Live/dead confocal microscopy images of g MG-63 cells and h MC3T3 cells on GG/GO-R scaffolds, GG/GO/Cur-R scaffolds, GG/GO-H scaffolds, and GG/GO/Cur-H scaffolds after 1, 3, and 5 days of cell seeding (Reproduced from [121], Copyright 2021, with permission from Elsevier) proposed dual-functional scaffolds presented a high potential for bone cancer treatment and recurrence prevention.
Zhu et al. [121] fabricated gellan gum/GO (GG/GO) scaffolds using an extrusion-based 3D printing system equipped with a 22G needle, nozzle temperature of 37°C, substrate temperature of 25°C and printing speed of 10 mm/s. Different filling patterns were considered, and the produced scaffolds were soaked in curcumin (Cur) solution to obtain GG/GO/Cur scaffolds. The produced GG/GO/Cur scaffolds could release 30% of the loaded Cur after 14 days in acidic medium (cancer environment). In vitro cell studies were conducted using mouse osteoblast MC3T3 cells and human osteosarcoma MG-63 cells. Based on the CCK-8 assay, the results showed that the fabricated GG/GO/Cur scaffolds could inhibit the viability of MG-63 cells by 50%, 82%, and 93% on day 1, 3, and 5, respectively, which were signifi-cantly lower compared to the GG/GO scaffolds (Fig. 2e). It was also possible to observe that MC3T3 attachment and proliferation on GG/GO/Cur scaffolds was significantly better than GG/GO scaffolds (Fig. 2f). The results were confirmed by live/dead confocal microscopy images of MG-63 cells and MC3T3 cells on the different scaffolds (Figs. 2g and 2h). No significant differences were observed regarding the different filling patterns.
Besides carbon nanomaterials, other materials like selenium have also been investigated for bone cancer applications. However, due to the relatively complex material preparation and characterization process, a limited number of publications have reported the use of selenium for 3D scaffolds. Most studies focused on the incorporation of selenium into ceramic, polymeric, and metallic nanoparticles, as well as a coating of metallic substrate [122][123][124][125][126][127][128][129][130][131][132][133][134][135][136][137]. These studies suggested that selenium is an effective functional material that can be used to treat bone cancer problems without affecting normal cells, also promoting bone regeneration, which is related to its antioxidant and prooxidant properties.
Scaffolds with advanced functional materials have great potential for bone cancer treatment and bone regeneration applications, not requiring additional assistance measures after clinical operation. Moreover, due to the use of biocompatible and biodegradable materials, no second operation is required, which accelerates recovery time. Through the development of novel materials and advanced manufacturing technologies, significant advances are expected in this field.

Drug-loaded scaffolds
Three-dimensional scaffolds have been proposed that are loaded with chemotherapeutics or anticancer drugs such as DOX, and they achieved promising therapeutic results. Chen et al. [138] prepared porous PCL scaffolds (5 mm in height and 10 mm in diameter) using a filament-based extrusion 3D printing machine, loaded the scaffolds by immersing them in a solution containing chitosan, nanoclay, and β-tricalcium phosphate (β-TCP), and freeze-dried the product. PCL was used to provide mechanical strength, while PCL/chitosan/nanoclay/β-TCP/DOX was applied for local sustained drug delivery and bone regeneration. Immortalized human bone marrow-derived mesenchymal stem cells (hBMSCs) were used for in vitro biological evaluation. The scanning electron microscopy (SEM) imaging, confocal microscopy imaging, and DNA quantification results showed that the produced scaffolds were able to support cell attachment, enabling high cell viability and good cell infiltration of hBMSCs. The osteoinductivity of the scaffolds was evaluated through alkaline phosphatase (ALP) and osteocalcin staining, and the results showed that cell mineralization started at day 7 after cell seeding. Moreover, the results of drug release kinetics based on DOX showed a fast release on scaffolds without nanoclay (94% of DOX was released within 4 days) compared to the scaffolds containing nanoclay (up to 47% of DOX was released after 56 days). Moreover, the release profile could be controlled by adjusting the amount of chitosan. The biological impact of the DOX release was not investigated by the authors, but as DOX is an anticancer drug, the results seemed to indicate a significant potential of these scaffolds for both cancer treatment and bone regeneration. This was confirmed by Ahangar et al. [22] using polymeric scaffolds loaded with DOX. These scaffolds were produced using a Flashforge Creator Pro 3D printing system (Flashforge, China) with PORO-LAY filaments (Lay-FOMM 40/60 and Gel Lay; Kai Parthy, Germany), including a nozzle diameter of 0.3 mm, printing temperature of 220°C, bed temperature of 50°C, and printing speed of 18 mm/s. DOX was loaded onto the scaffolds in a laminar flow safety cabinet. The produced scaffolds were evaluated in terms of morphology, drug-release kinetics, and biological performance. The results showed that 60-75% of the loaded DOX was released from the scaffolds after 7 days (Figs. 3a and 3b). Prostate cancer LAPC4 cells and patient-derived prostate cancer spine metastases cells were used for the in vitro biological evaluation. Both the Alamar blue and MTT assay results showed that the metabolic activity of LAPC4 cells was reduced after 7 days of treatment, and the Gel Lay scaffolds exhibited the lowest metabolic activity (Figs. 3c and 3d). In the case of patient-derived prostate cancer spine metastatic cells, the decrease in metabolism was more significant, which might be due to the increased sensitivity of these cells to DOX. Overall, the produced DOX-loaded scaffolds significantly reduced metabolic activity, proliferation, migration, and spheroid growth of both the LAPC4 cells and the patientderived prostate cancer spine metastases cells.
Lahr et al. [139] produced medical-grade PCL scaffolds using the melt-electrospinning technique and loaded the scaffolds with DOX by soaking them in a DOX solution. The in vitro drug release profiles showed that 72%, 80%, and 88% of the loaded DOX was released after two days from the scaffolds containing 0.4, 2, and 10 μg/mg DOX, respectively. In addition, the in vitro viability of Saos-2 cells decreased to (80 ± 7)%, (54 ± 6)%, and (27 ± 5)% after 2 days of treatment with the scaffolds containing 0.4, 2, and 10 μg/mg, respectively, and further decreased to (68 ± 3)%, (4 ± 2)%, and (1.2 ± 0.1)% after 5 days, respectively. In vivo biological studies were conducted using female NOD-SCID IL2Rγnull (NSG) mice xenografted with Saos-2 cells. Scaffolds loaded with 1%, 5%, and 10% of the administered intravenous dose of DOX (4 mg/kg body weight) were evaluated and compared with the direct intravenous administration group without using scaffolds. The results showed that the 10% scaffold group exhibited a significant decrease of cancer cell invasion and the increase in cancer cell death in comparison with the Fig. 3 a Release curve of LayFoMM 60 discs, LayFoMM 40 discs, and GelLay discs loaded with DOX over 7 days. b Total amount of DOX released from LayFoMM 60 discs, LayFoMM 40 discs, and Gel-Lay discs. Metabolic activity of LAPC4 cells treated with DOX-loaded LayFoMM 60 discs, LayFoMM 40 discs, and GelLay discs for 7 days, assessed through c Alamar blue assay and d MTT assay. * P < 0.05, ** P < 0.01, and ***P < 0.001 (Reproduced from [22], Copyright 2018, with permission from MDPI). e1 Ex vivo micro-CT reconstruction of the orthotopic niche in two different planes, showing the trabecular network (green), ramiform calcifications (brown), and the defect zone (yellow) containing the scaffolds loaded with DOX. e2 Lamin A/C staining at the region of the scaffolds loaded with DOX (yellow), showing the local invasion pattern of Saos-2 cells (brown) into the scaffolds loaded with 1%, 5%, and 10% of the administered intravenous DOX dose (4 mg/kg body weight). e3 Apoptotic assay results showing the induction of apoptosis (red) in mice treated with scaffolds loaded with 1%, 5%, and 10% of the administered intravenous DOX dose (4 mg/kg body weight). e4 Quantitative infiltration area and penetration depth analysis of Saos-2 cells in mice treated with scaffolds loaded with 1%, 5%, and 10% of the administered intravenous DOX dose (4 mg/kg body weight). f Hematoxylin-eosin (H&E) staining of the liver (the white arrows show central vein congestion and the black asterisks show multiple necrotic foci). Masson's trichrome and H&E staining of heart cross-sections through both ventriculi (the red box shows right ventricular dilation, the black arrows show vacuolization of cardiomyocytes) in mice treated with scaffolds loaded with 1%, 5%, and 10% of the administered intravenous DOX dose (4 mg/kg body weight) (Reproduced from [139], Copyright 2020, with permission from Elsevier) 1% and 5% groups (Fig. 3e). After 14 days of treatment, in contrast to the scaffold approach, the direct intravenous DOX administration presented side effects as observed by a significant body and organ weight reduction (Fig. 3f).
In addition to DOX, other chemotherapeutics such as 5-FU, ifosfamide, methotrexate, and cisplatin have been evaluated for bone cancer cells. Salmoria et al. [140] investigated polythene (PE)/5-FU waffles manufactured using laser-based powder bed fusion with different laser power densities. In this process, PE was mixed with 5-FU in a rotator for 10 min at 30 r/min and sintered with a CO 2 laser (9 W). The waffles were produced using a laser beam diameter of 250 μm, powder bed temperature of 45°C, laser scanning speed of 350 mm/s, layer thickness of 250 μm, hatch space of 125 μm, and laser power ranging between 3 and 5 W. The results showed that the waffles produced using 3 W presented lower flexu-ral modulus than those using 5 W due to a lower degree of sintering, and the PE/5-FU waffles presented lower flexural modulus than PE waffles. Similar results were observed for the fatigue strength. The drug release results showed an initial burst release of the loaded drug (50 mg/g) in the first 4 h, and a slower and constant rate in the following 2 days. The waffles produced using 3 W exhibited a faster drug release due to high porosity. Salmoria et al. [141] also fabricated implantable PCL/5-FU tablets using laser-based powder bed fusion with different values of laser power (3 and 7 W). The produced PCL/5-FU tablets also exhibited an initially rapid drug release and then a long-term slow release. More recently, the authors produced PE/5-FU intrauterine devices loaded with progesterone for ovarian cancer applications [142].
Wang et al. [143] fabricated poly-L-lactic acid (PLLA) cylindrical and spherical implants using a vat photopolymer-ization system. The produced PLLA implants were soaked in dichloromethane solution containing DOX, ifosfamide, methotrexate, and cisplatin. The results showed that the spherical implants had higher drug loading efficiency than the cylindrical ones, suggesting that the shape of the implant affects the drug encapsulation efficiency. In vivo studies were also conducted using nude mice xenografted with human osteosarcoma U 2 OS cells. The drug-loaded PLLA implants were implanted after tumor resection and the treatment effect was compared with the intraperitoneal drug injection group. The implanted drug-loaded implants could sustain the release of methotrexate, DOX, ifosfamide, and cisplatin for up to 12 weeks, and no tumor recurrences were observed.
Very few studies have reported on the use of drug-loaded scaffolds to treat bone cancer. However, a number of papers focused on different cancer types presenting high metastasis risk to bone tissue, and thus they are considered relevant within the scope of this review. Yi et al. [144] investigated the use of 3D-printed poly(lactide-co-glycolide)/PCL/5-FU (PLGA/PCL/5-FU) patches as anticancer drug delivery systems (Figs. 4a and 4b). Different concentrations of 5-FU (1, 5, 10, and 15 wt%, named as P10, P50, P100, and P150) were incorporated into PCL/PLGA blends (at a ratio of 1:1) and processed using an extrusion-based multi-head deposition system under a pressure of 600 kPa and a temperature of 140°C. The patches were produced using different layers (2, 4, and 8) and fiber deposition layouts (latticed, slant, and triangular pattern), allowing for different drug release rates due to differences in the surface area. The results showed that both slanted and triangular patches presented slower 5-FU release rates than the lattice patches. Moreover, it was possible to observe that the patches with the highest 5-FU concentration (15 wt%) released 30% of the loaded drug after 4 weeks. The in vitro biological experiment showed that all produced patches reduced the proliferation of human pancreatic cancer MIA PaCa-2 cells to less than 50%, while maintaining that of hADSCs over 50%, indicating that the produced patches were more effective on cancer cells than healthy cells. The results of in vivo tests conducted on male Balb/c nude mice xenografted with MIA PaCa-2 cells suggested that the 10 wt% patches presented a better inhibition effect on cancer cells and less toxicity on healthy cells. Additionally, no mouse died during the experiment, indicating the low systemic toxicity on internal organs. These results seemed to prove that the 3D-printed drug-loaded patches facilitate local drug delivery with limited side effects. Yang et al. [145] produced PLGA scaffolds containing 5-FU and NVP-BEZ235 (PFN scaffolds) using a near-field electrospinning approach with a flow rate of 0.16 mL/h, 2.8 kV voltage and 3 mm distance between the printing needle and the collector. The scaffolds presented different average pore sizes (50, 100, 150, and 200 μm) and drug loads (6% of 5-FU and 0.005% of NVP). The drug release kinetics results showed that both 5-FU and NVP had a fast release in the first 7 days. It was also observed that the release rate increases by enlarging the pore size. In vitro biological studies were conducted using the MTT assay with human breast cancer MDA-MB-231 cells and NIH 3T3 fibroblasts. The anti-tumor activity of the PFN scaffolds was compared with free drugs (5-FU, NVP), dual drugs (5-FU + NVP), and control groups. After 4 days of treatment, the dual drug group exhibited lower cell viability than the other groups. After 7 days, the PFN scaffolds inhibited 37.83% of MDA-MB-231 cells, which was higher than the control (2.37%), 5-FU (15.19%), NVP (7.66%), and dual drug (21.47%) groups. The results also showed that the PFN scaffolds did not affect the NIH 3T3 fibroblasts' cell growth. These findings were confirmed by both confocal microscopy and Annexin V-PE/7-AAD cell apoptosis assay. The in vivo anticancer properties of PFN scaffolds were evaluated on MDA-MB-231-bearing mice. After 4 weeks of therapy, the tumor volumes were significantly smaller (< 600 mm 3 ) in the case of PFN scaffolds than the tumor volumes in the dual drug (900 mm 3 ), control, and single drug groups (both around 1400 mm 3 ) (Figs. 4c-4g). The H&E staining imaging results suggested that the PFN scaffolds caused necrosis within the tumor tissues and reduced metastases in the lungs, while metastases were detected in the other groups. All of the results seemed to suggest that PFN scaffolds correspond to a viable approach to treat tumors with negligible side effects on other organs. Chou et al. [146] used an extrusion-based 3D printer (Hyrel system 30 M) to produce nanogel discs containing PTX and rapamycin. Briefly, solgels were prepared by mixing poloxamer, PTX, rapamycin, and tert-butanol, and were printed at a printing speed of 10 mm/s. Finally, the printed discs were dehydrated in an oven at 37°C for 24 h. The drug release results showed that the discs presented slower release profiles than sol-gels and that the release of PTX was also slower than rapamycin. The relevant in vivo studies were based on female athymic NCr-nu/nu mice xenografted with ES-2-luc cells, and the results showed that the drug-loaded discs could successfully deliver the drugs peritoneally, reducing both the risk of postsurgical peritoneal adhesion and the progression of tumor, thus increasing the survival rate. Dang et al. [147] used an extrusion-based additive manufacturing system to produce PCL scaffolds (diameter of 6 mm and height of 0.75 mm) and loaded with DOX. Female NOD-SCID IL2Rγnull (NSG) mice xenografted with MDA-MB-231 cells were used for their in vivo studies (Fig. 4h), and the results showed that the DOX-loaded PCL scaffolds delayed the growth and recurrence of cancer (Figs. 4i and 4j). The produced scaffolds also reduced the cancer metastases in the lungs, liver, and spleen.
Metal structures have also been investigated as local drug delivery systems. Aiming to develop bone implants with improved biointegration and anticancer properties, Maher , and layers (2, 4, and 8, scale bar 2 mm). b SEM images of the surface of P0, P10, P50, P100, and P150 patches. Scale bar 500 μm (Reproduced from [144], Copyright 2016, with permission from Elsevier). c Photographs and in vivo fluorescence images of mice after 4 weeks of treatment in the control group, 5-FU group, NVP group, 5-FU + NVP group, and PFN group. d Body weight changes of mice in the control group, 5-FU group, NVP group, 5-FU + NVP group, and PFN group. e Tumor volume changes of mice in the control group, 5-FU group, NVP group, 5-FU + NVP group, and PFN group. f Photographs of tumors collected from mice under different treatment approaches after 4 weeks of treatment. f1 Control, f2 5-FU, f3 NVP, f4 dual drugs (5-FU/NVP), f5 PFN scaffolds. g Survival curves of mice in the control group, 5-FU group, NVP group, 5-FU + NVP group, and PFN group within 60 days (Reproduced from [145], Copyright 2020, with permission from Elsevier). h Workflow of in vivo experiments. i Bright-field and j H&E staining images of the recurrent tumors at the primary tumor site in the control group, I.V. group, low-dose group, and high-dose group (Reproduced from [147], Copyright 2020, with permission from John Wiley and Sons) et al. [148] used a laser-based powder bed fusion system equipped with 300 W laser (1070 nm) to produce Ti6Al4V wafers under an inert argon atmosphere. The produced wafers were submitted to an anodization process to create a surface topography suitable for bone applications, and they were finally loaded with DOX and apoptosis-inducing ligand (Apo2L/TRAIL) under vacuum. The in vitro results showed that, after 24 h of treatment, the anticancer drugloaded wafers were able to significantly reduce the viability of MDA-MB-231-TXSA cells, indicating that it can be a viable approach to treat bone cancers.
Drug-loaded scaffolds have shown good bone cancer treatment efficacy with very limited side effects due to the localized drug delivery mechanism. For this approach, how-ever, the sustained and controllable release of drugs is critical. The existing studies have been limited to qualitative research, hence more comprehensive investigations on precise drugrelease rates are needed.

Scaffolds for photothermal/magnetothermal therapy
As cancer cells are temperature sensitive, the use of scaffolds containing photo-sensitizers or magneto-sensitizers seems to represent a feasible alternative to treat bone cancers. These scaffolds generate heat when irradiated with NIR or exposed to a magnetic field. Zhang et al. [149] used pressure-assisted extrusion-based 3D printing systems to produce PCL and mesoporous bioactive glass (MBG, Si/Ca/P molar ratio of 80/15/5) composite scaffolds containing different concentrations of Fe 3 O 4 (0, 5, 10, and 15 wt%) (Figs. 5a and 5b). Briefly, MBG powders and magnetic Fe 3 O 4 nanoparticles were mixed and added into a PCL solution (the weight ratio between Fe 3 O 4 /MBG and PCL was 62:38), forming a paste that was then 3D-printed (nozzle diameter of 0.25 mm, pressure ranged between 1.5-3.0 bar, and printing speed ranged between 1.5 and 5 mm/s), producing scaffolds with uniform macropores (400 μm), high porosity (60%), and compressive strength varying between 13-16 MPa. Under an alternative magnetic field (strength of 180 G and frequency of 409 kHz), the temperature of the 15 wt% Fe 3 O 4 /MBG/PCL scaffolds rapidly increased from 20 to 43°C within 2 min. The results also showed that the rate of temperature increase rises by elevating the Fe 3 O 4 concentration. Moreover, the scaffolds were loaded with DOX, and drug release studies showed that around 30% of the loaded DOX was released on the first day. Despite the potential of these scaffolds for cancer applications, the authors did not perform any biological studies to evaluate the concept. Their biological studies were limited to in vitro proliferation and differentiation assays using primary hBMSCs without applying any magnetic field. The results showed that the addition of magnetic  (Figs. 5c-5f). Moreover, no significant pathological toxicity was observed in the major organs (heart, lung, spleen, liver, and kidney) in any of the groups. The inhibition effect was attributed to the Fenton-like effect, a reaction between Fe 3 O 4 and CaO 2 generating OH and increasing the reactive oxygen species (ROS) level, which induces the termination of cancer. Moreover, micro-CT and CLSM images showed good in vivo osteogenesis capability of AKT-Fe 3 O 4 -CaO 2 scaffolds based on a male Sprague-Dawley rat model.
Wang et al. [151] combined additive manufacturing with a hydrothermal method for the in situ growth of molybdenum disulfide (MoS 2 ) nanosheets on the strut surface of AKT scaffolds (MS-AKT scaffolds). Under NIR irradiation (0.5 W/cm 2 ), it was possible to observe that the MS-AKT scaffolds could rapidly reach 115°C after 5 min of irradiation under a dry environment (air), and 50°C after 10 min under wet conditions (phosphate-buffered saline). In vitro cell studies were conducted using Saos-2, MDA-MB-231 cells, and rabbit BMSCs, and the results showed that, after submitting the MS-AKT scaffolds to a cycle of three irradiation stages (0.6 W/cm 2 , 10 min), the viability of MDA-MB-231 cells was reduced to around 5%. Saos-2 cells were also inhibited. Without the irradiation, MS-AKT scaffolds were able to promote rabbit BMSCs proliferation and upregulate the expression of osteogenic genes (Runx2, OCN, OPN and ALP). The in vivo biological studies were based on Balb/c mice xenografted with Saos-2 cells (Figs. 5g-5i). In the case of combining MS-AKT scaffolds with irradiation (0.5 W/cm 2 , 10 min, every 2 days) almost no tumors were observed after 14 days, a value significantly lower when compared to the other considered cases (Fig. 5j). Zhuang et al. [152] prepared ceramic inks containing AKT and Fe (Figs. 5k-5m) to produce 3D-printed scaffolds using an extrusion-based system. The authors conducted in vitro studies using murine osteosarcoma LM-8 cells and considered three conditions: magnetic field (896.8 A/m) only; NIR (0.70 W/cm 2 ) only and a combination of magnetic field and NIR. The findings revealed that cell viability decreased to 59.2% in the case of magnetic field, to 81.6% in the case of NIR, and to 2.0% in the combined case. The Copyright 2019, with permission from John Wiley and Sons). g Thermal images and h temperature changes of tumor-bearing mice after being implanted with MS-AKT and AKT scaffolds under NIR irradiation for up to 10 min. i Whole-body fluorescence imaging of mice after 0 day (left) and 7 days (right) of treatment with MS-AKT scaffolds with and without irradiation, and with AKT scaffolds with and without irradiation. j Relative tumor volume of mice treated with MS-AKT scaffolds with irradiation, MS-AKT scaffolds without irradiation, AKT scaffolds with irradiation, and AKT scaffolds without irradiation (Reproduced from [151], Copyright 2017, with permission from Springer Nature). k Photographs and (l and m) SEM images of (k1, l1, and m1) AKT scaffolds, (k2, l2, and m2) 1Fe-AKT scaffolds, (k3, l3, and m3) 2Fe-AKT scaffolds, and (k4, l4, and m4) 3Fe-AKT scaffolds (Reproduced from [152], Copyright 2019, with permission from American Chemical Society) Fe-AKT scaffolds also showed good cell adhesion, proliferation, and osteogenic properties (high expression levels of RUNX2, OPN, OCN, and BMP2 genes) on rabbit BMSCs.
Further materials such as bioglass (BG) have also been used to fabricate photothermal/magnetothermal scaffolds. Yang et al. [153] prepared BG scaffolds using a pressureassisted extrusion system, which were then soaked in a solution containing mesoporous silica-coated 2D Nb 2 C MXene and S-nitrosothiol to produce MS/MXene-BG-SNO scaffolds (shortly referred to as MBS scaffolds). Once irradiated under NIR (1064 nm, 1.0 W/cm 2 ) for 10 min, the temperature rapidly increased to 52°C. A correlation between temperature increase and light intensity was also established. In vitro studies were conducted using Saos-2 cells, and the results showed that after irradiation for 10 min (1.0 W/cm 2 ), the viability of Saos-2 cells decreased to 25-35% and further decreased to 20-30% by increasing the irradiation time to 30 min. In vivo cell studies were conducted using female Balb/c nude mice xenografted with Saos-2 cells. In this case, MBS scaffolds were compared to BG scaffolds loaded with Nb 2 C (NBGS) considering stages of irradiation for 10 min and NIR intensity of 1.0 W/cm 2 every 2 days. The results showed that both NBGS and MBS eliminated cancer without recurrence after 2 weeks of treatment due to the precise control of nitric oxide release and the anticancer effects of nitric oxide. Moreover, the MBS group eliminated cancer faster than the NBGS group, as observed from the staining results showing more apoptotic cells. No significant body weight change and metastasis were observed in other major organs (heart, liver, spleen, lung, and kidney). Moreover, the in vivo studies involving on male Sprague-Dawley rats showed that the MBS scaffolds exhibited good osteogenic and angiogenic properties. Similar results were obtained by Pan et al. [154] using BG scaffolds loaded with Ti 3 C 2 on female Balb/c nude mice xenografted with Saos-2 cells (Fig. 6a) and Sprague-Dawley (SD) rats. The in vitro results revealed good cancer treatment effects and demonstrated that the fabricated scaffolds can support the proliferation and differentiation of human marrow-derived BMSCs, exhibiting enhanced osteogenic capability. The in vivo results also confirmed the output of in vitro cancer treatment. Additionally, the osteogenesis rate of the Ti 3 C 2 -loaded scaffold was 2.5 times higher than that of BG scaffolds. Wang et al. [155] used BG scaffolds coated with molybdenum disulfide Fig. 6 a H&E staining of the major organs of Saos-2-bearing mice on day 14 with BGS scaffolds with and without irradiation, and with TBGS scaffolds with and without irradiation. Scale bars 100 μm (Reproduced from [154], Copyright 2019, with permission from John Wiley and Sons). b Relative cell viability and tumor volume in the control, BG scaffolds with irradiation, BGM0.3 scaffolds with irradiation, BGM0.5 scaffolds with irradiation, and BGM1 scaffolds with irradiation at day 14 (Reproduced from [155], Copyright 2020, with permission from Elsevier). c1-c5 Photographs of c1 5Cu-BGC, c2 5Fe-BGC, c3 5Mn-BGC, c4 5Co-BGC, and c5 BGC scaffolds. d Temperature changes of 5Cu-BGC, 5Fe-BGC, 5Mn-BGC, 5Co-BGC, and BGC scaffolds under irradiation. e Whole-body fluorescence images of tumor in mice treated with 5Cu-BGC scaffolds with irradiation, 5Fe-BGC scaffolds with irradiation, 5Mn-BGC scaffolds with irradiation, 5Co-BGC scaffolds with irradiation, and BGC scaffolds with irradiation at day 0 and day 14.
f Tumor volume changes of mice with 5Cu-BGC scaffolds with and without irradiation, 5Fe-BGC scaffolds with and without irradiation, 5Mn-BGC scaffolds with and without irradiation, 5Co-BGC scaffolds with and without irradiation, and BGC scaffolds without irradiation. g Photographs of tumors obtained from different mice treated with 5Cu-BGC, 5Fe-BGC, 5Mn-BGC, 5Co-BGC, and BGC scaffolds with irradiation on day 14 (Reproduced from [158], Copyright 2018, with permission from Elsevier). h Schematic illustration of the fabrication process and functions of larnite/C scaffolds (Reproduced from [160], Copyright 2020, with permission from Elsevier). i Whole-body bioluminescent imaging of tumor in mice treated with BC scaffolds with and without irradiation, and DOPA-BC scaffolds with and without irradiation (Reproduced from [161], Copyright 2016, with permission from Elsevier) (MoS 2 ) on nude rats xenografted with MNNG/HOS cells (Fig. 6b) and SD rats. The in vitro results showed that the produced scaffolds coated with MoS 2 presented a good ability for both cancer treatment and rabbit BMSCs mineralization. The in vivo biological studies confirmed these results in terms of both tumor inhibition and bone regeneration. Dang et al. [156] used BG scaffolds functionalized by CuFeSe 2 nanocrystals and prepared by 3D printing and solvothermal synthesis on nude mice xenografted with Saos-2 cells and a New Zealand white rabbit model. Like in previously reviewed cases, both the in vitro and in vivo results confirmed good cancer treatment and bone tissue regeneration ability. Similar findings were obtained using 3D-printed BG scaffolds loaded with DOX on female nude mice xenografted with K7M2wt cells [157].
The effect of the element composition was investigated by Liu et al. [158], wherein a commercially available BG (CaSiO 3 ) was modified by incorporating different levels of Cu, Fe, Mn, and Co (Cu(NO 3 ) 2 ·4H 2 O, Fe(NO 3 ) 3 ·9H 2 O, Mn(NO 3 ) 2 ·4H 2 O and Co(NO 3 ) 2 ·6H 2 O) through a sol-gel method using tetraethyl orthosilicate and triethyl phosphate (Fig. 6c). The sol-gel derived powders were sintered at 800°C for 3 h, mixed with a Pluronic F-127 solution, and printed using an extrusion-based system. The scaffolds containing Cu exhibited the fastest degradation rate (21.3% weight loss after 28 days) and the best photothermal performance (wavelength of 808 nm and light intensity of 0.48 W/cm 2 ). The in vivo anticancer properties of the produced scaffolds were evaluated based on nude mice xenografted with Saos-2 cells. The results showed that the scaffolds containing Cu under NIR irradiation (0.75 W/cm 2 for 15 min, every 2 days) presented the highest cancer inhibition effect (tumor tissue necrosis rate of 94.9%) compared with all other groups (Figs. 6d-6g). Moreover, scaffolds containing Fe and Mn promoted the attachment and proliferation of rabbit BMSCs, scaffolds containing Co had the highest expression level of ALP, and both BG and Cu scaffolds showed the strongest mineralization ability. Similar results were reported by Ma et al. [159], who prepared a mixture of Fe and CaSiO 3 powders to produce scaffolds using a laser-based powder bed fusion system under inert conditions. The produced Fe-CaSiO3 scaffolds showed anticancer characteristics both in vitro (under NIR irradiation of 0.6 W/cm 2 for 15 min, every 2 days) and in vivo (under NIR irradiation of 0.8 W /m 2 for 10 min, every 2 days), as well as osteogenesis properties when used without irradiation. Moreover, due to the release of Fe ions during treatment and its interaction with H 2 O 2 inside the tumor, ROS generation was induced, enhancing the tumor therapeutic effect of the scaffolds due to the synergistic photothermal and ROS effects.
Fu et al. [160] fabricated porous free carbon-embedding larnite (larnite/C) scaffolds using a pressure-assisted extrusion-based system. Prior to printing, CaCO 3 powders were mixed with silicone resin in isopropyl alcohol (Fig. 6h). The printed scaffolds were heated (rate of 2°C/min for 3 h) in an argon atmosphere up to 1200°C. Larnite scaffolds, fabricated using sol-gel larnite powders followed by sintering, were used as control for the biological studies. The results showed that the temperature of the larnite/C scaffolds rapidly increased to 61-63°C (dry conditions) and 46-48°C (wet conditions) under irradiation (808 nm, 0.75 W/cm 2 ) for 5 min. The in vitro cell studies therein were conducted using MNNG/HOS cells and rat BMSCs on both larnite and larnite/C-3 scaffolds. The CCK-8 assay results showed that, by increasing the NIR intensity from 0.5 to 1.25 W/cm 2 , the viability of MNNG/HOS cells decreased from (70 ± 5)% to (5 ± 2)%. Without irradiation, the larnite/C scaffolds presented better osteogenic results, as confirmed by high rat BMSCs viability and high expression levels of osteogenic markers such as ALP, OCN and Runx-2. The in vivo studies were conducted based on nude mice xenografted with MNNG/HOS cells, and the results showed that the combined use of larnite/C scaffolds and irradiation (0.75 W/cm 2 for 10 min, every 2 days) significantly decreased the tumor volume. In addition, substantial necrosis of the tumor tissue was observed by H&E staining imaging. The reconstructed micro-CT images showed that larnite/C-3 scaffolds resulted in higher bone mineral density (0.41 g/cm 3 ) and bone volume/tissue volume ratio (40%) compared to larnite scaffolds (0.3 g/cm 3 and 35%).
Dopamine, commonly used to improve the biological performance of scaffolds, has been used to increase the scaffold temperature upon NIR irradiation, thus improving its anti-cancer capabilities. Ma et al. [161] used an extrusion-based system to produce Ca 7 Si 2 P 2 O 16 scaffolds. The produced bioceramic scaffolds were soaked in a polydopamine solution and dried to obtain the final polydopamine-bioceramic (DOPA-BC) scaffolds. Upon irradiation with NIR (808 nm, 0.34 W/cm 2 ), the temperature of DOPA-BC scaffolds increased to 92°C in a dry environment and to 50°C in a wet environment. Saos-2 and MDA-MB-231 cells were used to evaluate the in vitro photothermal effect of the DOPA-BC scaffolds. The results showed that under irradiation (0.38 W/cm 2 , 10 min), the viability of Saos2 cells and MDA-MB-231 cells decreased to around 99.2% and 97.4%, respectively. In vivo cancer studies were conducted based on female nude mice xenografted with MDA-MB-231 cells both under irradiation (0.38 W/cm 2 for 10 min, every 2 days) and no irradiation. In the case of irradiation, the temperature was observed to rapidly increase to values higher than 50°C, and the weight of the tumor was significantly reduced (Fig. 6i). In the case of the hyperthermic central area (temperatures above 50°C), the cancer cells underwent coagulative necrosis caused by cell membrane collapse, protein denaturation, termination of enzyme activity, and mitochondrial dysfunction. In the case of the hyperthermic peripheral region (temperatures above 45°C), the ablation of tumors was triggered by cancer cell apoptosis. Moreover, histological analysis based on the New Zealand white rabbit model showed that, after 8 weeks of implantation without NIR irradiation, the bone regeneration was stimulated.
Ma et al. [162] investigated the use of GO-modified β-TCP (GO-TCP) scaffolds for bone cancer applications (Figs. 7a and 7b). The TCP powder was mixed with a polyvinyl alcohol (PVA) solution and printed using an extrusion-based machine. Finally, the scaffolds were soaked in a solution containing GO. After NIR irradiation (0.36 W/cm 2 ) for 10 min, the temperature increased to 71-85°C at dry conditions and to 39-45°C at wet conditions, depending on the GO concentration increased from 0.25 to 1 mg/mL. In vitro biological studies were conducted using MG-63 cells and rabbit BMSCs. Under irradiation (0.36 W/cm 2 for 10 min), the GO-TCP scaffolds inhibited 80% of the MG-63 cells, and the cell viability further decreased by 92.6% under a longer irradiation time (30 min). The results also showed that rabbit BMSCs seeded on irradiated GO-TCP scaffolds presented high ALP activity and bone-related gene expression levels (Runx2, OCN, and BSP). In vivo studies were conducted based on nude mice xenografted with Saos-2 cells and the New Zealand white rabbit model considering four different cases: TCP scaffolds with irradiation (0.42 W/cm 2 for 10 min, every 2 days), TCP scaffolds without irradiation, GO-TCP scaffolds with irradiation (0.42 W/cm 2 for 10 min, every 2 days), and GO-TCP scaffolds without irradiation. The findings revealed that the necrosis rate of cancer cells reached up to 83.28% in the GO-TCP scaffolds with NIR irra- Fig. 7 a Schematic illustration of the bifunctional GO-TCP scaffold concept and the in vivo experimental procedure. b1-b3 Photographs of β-TCP (white) and GO-TCP (black) scaffolds (Reproduced from [162], Copyright 2016, with permission from John Wiley and Sons). c Photographs of tumors excised after 14 days of treatment using β-TCP scaffolds with and without irradiation, and β-TCP-C scaffolds with and without irradiation (Reproduced from [163], Copyright 2020, with permission from Royal Society of Chemistry). d1 Viability of Saos-2 cells on TCP scaffolds with and without irradiation, TCP-PDLLA scaffolds with and without irradiation, and TCP-PDLLA-5LB scaffolds with and without irradiation. d2 TCP-PDLLA-5LB scaffolds irradiated under different NIR light intensities. d3 TCP-PDLLA-5LB scaffolds irradiated under NIR under different irradiation times. e1-e6 SEM images of Saos-2 cells on TCP (e1, e2), TCP-PDLLA (e3, e4), TCP-PDLLA-5LB (e5, e6) scaffolds without (e1, e3, e5) or with (e2, e4, e6) NIR irradiation. Thermal images of tumor-bearing mice after being implanted with (f1) TCP, (f2) TCP-PDLLA, and (f3) TCP-PDLLA-5LB scaffolds under NIR irradiation. Micro-CT images of TCP (g1, g2), TCP-PDLLA (g3, g4), and TCP-PDLLA-5LB (g5, g6) scaffolds implanted into criticalsized femoral defects of rabbits after 8 weeks (green for scaffolds and red for bone-related tissues) (Reproduced from [164], Copyright 2019, with permission from Elsevier) diation, which was significantly higher than the other groups. Moreover, the histomorphometric measurements revealed improved osteogenic differentiation of rabbit BMSCs in the GO-TCP scaffolds without irradiation due to the specific bioactive groups (carboxyl and hydroxyl groups) and protein absorbance of GO. Dong et al. [163] studied the use of carbon aerogel (CA)-coated β-TCP (TCP-CA) scaffolds. An ink prepared by mixing TCP powder in a PVA solution was printed using an extrusion-based additive manufacturing system, producing scaffolds that were then coated by immersing the TCP scaffolds in a CA solution. Under NIR irradiation (1.0 W/cm 2 ) for 10 min, the temperature of the TCP-CA scaffolds increased to 74°C, while the temperature of the non-coated TCP scaffolds increased to 28°C. The associated in vitro studies conducted with MNNG/HOS cells under the same irradiation conditions showed that the cell viability significantly decreased in the TCP-CA group in comparison with the TCP group. It was also observed that, without irradiation, the TCP-CA scaffolds enhanced the attachment of bone marrow stromal cells, regulating the subsequent cellular behaviors. The scaffold groups evaluated in the in vitro studies were also considered in the in vivo studies conducted using Balb/c nude mice xenografted with MNNG/HOS cells. The results showed that the use of TCP-CA scaffolds with irradiation (1.0 W/cm 2 , keeping 52°C for 2 min, every 2 days) allowed for a cancer inhibition rate of 81.85%, which was higher than any other cases (Fig. 7c). Moreover, it was observed that TCP-CA scaffolds without irradiation could support in vivo osteogenesis. Dang et al. [164] also used TCP powder in a pluronic F-127 solution to produce scaffolds in an extrusion-based system. Scaffolds were then soaked in a solution containing lanthanum hexaboride (LaB 6 ) and poly(D,L-Lactic Acid) (PDLLA) at different concentrations (0.01, 0.03, and 0.05 g/mL corresponding to TCP-PDLLA-1LaB 6 , TCP-PDLLA-3LaB 6 , TCP-PDLLA-5LaB 6 , respectively). The results showed that the addition of PDLLA and LaB 6 increased the compressive strength of the scaffolds. Further results showed that, upon irradiating the scaffolds with a NIR laser (808 nm, 0.6 W/cm 2 ), the temperature of the scaffolds reached 100.7, 119.2, and 128.8°C under dry conditions (temperatures corresponding to the TCP-PDLLA-1LB, TCP-PDLLA-3LB, TCP-PDLLA-5LB groups, respectively) and 52.3, 62.4, and 66.3°C, respectively, under wet conditions. Saos-2 cells and rabbit bone marrow stromal cells were used for the in vitro studies and the scaffolds were irradiated under different light intensities (0.3, 0.4, 0.5, 0.6, and 0.7 W/cm 2 ) for 5, 10, 15, and 20 min. The results showed that cell viability decreased by increasing the NIR intensity and irradiation time (Fig. 7d). For the same NIR intensity and irradiation time, a significant decrease in cell viability was achieved in the TCP-PDLLA-5LB group (Figs. 7d and 7e). The in vitro studies conducted without irradiation demonstrated that the TCP-PDLLA-5LB scaffolds could support the attachment and growth of rabbit bone marrow stromal cells, with higher expression levels of osteogenic-related genes RUNX2, COL 1 and BMP2. In vivo studies were conducted based on nude mice xenografted with Saos-2 cells and a New Zealand white rabbit model (Figs. 7f and 7g). The H&E staining results showed that the NIR-irradiated (0.7 W/cm 2 for 10 min, every day) TCP-PDLLA-5LB scaffolds could inhibit tumor growth, and also support bone-forming activity in vivo, as indicated by micro-CT scanning results (Fig. 7g).
Photothermal/magnetothermal therapy has been widely investigated and subjected to a large number of in vitro and in vivo biological studies [149][150][151][152][153][154][155][156][157][158][159][160][161][162][163][164]. Positive results were observed not only in terms of bone cancer treatment but also for bone regeneration. However, these strategies need external assistance such as controllable NIR or magnetic field, which potentially increases the difficulty of the surgical procedure. Moreover, similar to the drug-loaded scaffold approach, these studies focused only on qualitative research. Thus, more comprehensive studies are needed on mathematical models to describe the materials, heat-release, and anticancer effects.

Conclusions and future perspectives
The current clinical approaches for bone cancer include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, all presenting certain limitations such as pain, allergies, low immunity, superinfections, and amputations. To address these limitations, novel therapeutic approaches are required. Recent developments in terms of materials and fabrication strategies opened the door to the development of novel and promising strategies, which have been discussed in detail in this review. Additive manufacturing processes-mainly extrusion-based, powder bed fusion, and vat photopolymerization techniques-have been employed to process a wide range of materials (e.g., bioceramics, metals, and polymers) and produce synthetic implants for postoperative applications (Table 4). Different approaches were proposed focusing on the development of scaffolds made with advanced functional materials, anticancer drug-delivery scaffolds, and scaffolds for photothermal and magnetothermal applications. The design and fabrication of dual-functional scaffolds, discussed in this review in detail, represents an emerging area. Such scaffolds are designed to fulfil an initial treatment function, which can be achieved by incorporating specific drugs and materials exhibiting cytotoxicity at high concentrations or by applying NIR irradiation or a magnetic field that significantly increases the temperature in a localized region. After this initial therapeutic stage, the scaffold must be able to induce bone regeneration. Despite the initial stage of research, the  [138,144,146,147] Laser-based powder bed fusion [140][141][142]148] Vat photopolymerization [143] Melt-electrospinning [139] Near-field electrospinning [145] Scaffolds for photothermal/magnetothermal therapy Extrusion-based additive manufacturing [149][150][151][152][153][154][155][156][157][158][160][161][162][163][164] Laser-based powder bed fusion [159] results reported in this paper are highly promising, suggesting that this could be a highly relevant approach for bone cancer treatment applications. All of the reported approaches have shown great inhibition effects on both bone cancer cells (e.g. The reported biological studies (both in vitro and in vivo) correspond to short-term studies, which are insufficient to fully understand the behavior of these scaffolds. Therefore, long-term experiments are required to understand the effect of some reinforced materials such as graphene and GO, cancer recurrence, and the secondary effects associated with the incorporated drugs, degraded products, and ion release. Contrary to current studies that are limited to mice and rabbit models, large animal models should be implemented before human trials can commence.
As bone is a load-bearing tissue, the mechanical properties of the produced scaffolds are highly relevant to recovery. This is particularly critical for dual-functional scaffolds, which must support physiological loads during a significant period, gradually transferring the loads to the new bone formed during the regeneration phase. However, the commonly used materials present significant drawbacks regarding their mechanical properties. Polymers and ceramic materials present poor mechanical properties compared to bone tissue, while metals exhibit strong mechanical properties leading to stress shielding problems [165,166]. Table 5 summarizes some of the inherent problems of the materials generally used in this field. Novel composite materials incorporating polymers, ceramics and low proportions of biodegradable metals (e.g., iron and zinc) should be investigated in the future to address these limitations. The performance of the scaffolds discussed in this review is strongly linked to the addition of functional materials, loaded drugs, and photo-sensitizers or magneto-sensitizers. For local cancer treatment implants, the sustained and controllable release of drugs is crucial. However, it is challenging to precisely control the drug release kinetics using the approaches proposed to date [172]. In the case of scaffolds for photothermal/magnetothermal therapy, there is no systematic study or mathematical model correlating the materials (and material properties), heat release, and anticancer effects. These models are vital to design optimized scaffolds, and also to numerically predict their behavior, thus minimizing the need for animal studies. It is also essential to create models that allow the prediction of the number of anticancer drugs or photo-sensitizers or magneto-sensitizers to be used for a specific stage of cancer development, which could contribute to the advancement of stratified/personalized cancer treatment approaches.
Author contributions YH was involved in conceptualization, investigation, writing-original draft, writing-review & editing, visualization; WW was involved in conceptualization, investigation, writing-review & editing, supervision, and funding acquisition; PB was involved in conceptualization, investigation, writing-review & editing, supervision, and funding acquisition.

Declarations
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human or animal subjects performed by any of the authors.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecomm ons.org/licenses/by/4.0/.